In eukaryotic cells calcium ions are the primary means of controlling protein-based molecular machines. The ability to record these calcium signals using fluorescent indictors has provided an entry into molecular mechanism and a means to follow subcellular signaling dynamics. Recent advances in genetically encoded calcium indicators (GECIs) now provide the ability to detect even small, transient, and rapidly fluctuating calcium signals within individual genetically-targeted cells in vivo. Calcium signaling regulates movement patterns that govern cell-cell contact essential for lymphocyte activation; other facets include close coupling to T cell receptor activation, reliance on Orai1, calcium- dependent transcriptional activation, and suppression of calcium signaling by regulatory T cells. There is a need to simultaneously track and read out calcium concentrations using endogenous transgenic expression of GECIs to (1) uncover mechanisms of regulatory T cell suppression, (2) visualize therapeutic modulation of endogenous T cell activation in disease models (3) identify endogenous polyclonal activation events in the lymph node for the first time. Moreover, the need for these capabilities are shared by neuroscientists seeking to measure, modify, and model brain circuit function for the President's Brain Initiative, the Brain Activity Map, and similar projects. We have created a novel; ratiometric GECI we call Salsa6f by fusing the green fluorescent indicator GCaMP6f with the red fluorescent protein tdTomato and have successfully tested it in vitro. This probe permits quantitative readout of Ca2+ concentrations and Ca2+-independent tracking. This proposal seeks to (1) generate a Cre-dependent GECI transgenic mouse strain with Salsa6f through site directed targeting of the ubiquitously expressed Rosa-26 locus, and (2) cross this line to Cre recombinase driver lines and validate Salsa6f function in immune cells, neurons, and glia. After crossing to drive expression in CD4+ T cells and Vav+ B cells, purified cells will be tested for Salsa6f indicator function, expression, and lack of interference in immune cell homing, localization, motility, proliferation, surface marker expression, and cytokine production, as measured in quantitative assays. Similar crosses will drive expression in CaMKII+ neurons and GFAP+ astrocytes. Brains sections from transgenic mice will be assessed for appropriate expression and neuronal and glial morphology. Neurons and glia will be cultured and the speed and sensitivity of Salsa6f measured upon field stimulation and during spontaneous activity. The new capabilities of Salsa6f transgenic mice will enable new approaches to identify, map, and relate the intricate molecular mechanisms and cell behaviors that collectively define immune and nervous system function.